PROJECT SUMMARY This application probes the role of the myosin motor protein in aging and in progressive, genetically based diseases of skeletal and cardiac muscles. We will employ a multidisciplinary approach that takes advantage of the powerful genetic tools available for Drosophila melanogaster along with a broad range of expertise that allows us to study myosin in an integrative manner, from its crystal structure and biochemical function through its effects upon muscle ultrastructure, fiber mechanical properties, cardiac physiology and locomotion. For Aim 1, we will examine the functional significance of specific residues within the skeletal muscle myosin motor and rod domains that are post-translationally modified during human aging. Using transgenic Drosophila, we will assess the effects of mutations at these sites on myosin ATPase activity, in vitro motility, thick filament formation and stability, indirect flight and jump muscle ultrastructure, fiber mechanics and organism locomotion. We will test the hypothesis that specific myosin amino acid residues that are subject to age-related post-translational modifications are critical for myosin's biochemical properties and structure and function of the myofibrillar apparatus. For Aim 2, we will examine how amino acid mutations associated with human age- related dilated cardiomyopathy affect the structure and biochemical properties of the myosin molecule, as well as the structure and physiological function of the Drosophila heart. We will gain mechanistic insights into protein structural features that are vital for cardiac myosin function using a unique in vivo method to produce mutant forms of the myosin protein for crystallography. Further, we will assess the power of the Drosophila system as a screening tool for identifying putative dilated cardiomyopathy myosin alleles defined in humans. This combined protein structural and in vivo screening approach will test the hypothesis that mutations that cause human dilated cardiomyopathy disrupt intramolecular communication leading to depressed myosin motor function, Drosophila cardiac dilation, heart wall thinning and reduced cardiac output. For Aim 3, we will define the roles of specific chaperone-affiliated proteins in inclusion body myopathy type 3, a progressive skeletal muscle disorder. Specific small heat shock proteins, a mitochondrial chaperone and an ubiquitin ligase anomalously accumulate in aggregates in our Drosophila model of this disease. Up- or down-regulating their expression, followed by structural and functional analyses of skeletal muscles, will yield insights into their roles in aging and in degenerative muscle disease. We will test the hypothesis that manipulating the levels of specific small heat shock proteins, a mitochondrial chaperone and an ubiquitin ligase can ameliorate or exacerbate progressive myosin-based myopathy phenotypes. Overall, our project will take advantage of an innovative experimental system to test significant questions regarding basic myosin function during aging of healthy and diseased skeletal and cardiac muscles.